MiR-221 Influences Effector Functions and Actin Cytoskeleton ...

MiR-221 Influences Effector Functions and ActinCytoskeleton in Mast CellsRamon J. Mayoral1., Lorenzo Deho1., Nicole Rusca1., Nenad Bartonicek2, Harpreet Kaur Saini2, Anton J.

Enright2, Silvia Monticelli1*

1 Institute for Research in Biomedicine, Bellinzona, Switzerland, 2 EMBL - European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United

Kingdom

Abstract

Mast cells have essential effector and immunoregulatory functions in IgE-associated allergic disorders and certain innateand adaptive immune responses, but the role of miRNAs in regulating mast cell functions is almost completely unexplored.To examine the role of the activation-induced miRNA miR-221 in mouse mast cells, we developed robust lentiviral systemsfor miRNA overexpression and depletion. While miR-221 favored mast cell adhesion and migration towards SCF or antigenin trans-well migration assays, as well as cytokine production and degranulation in response to IgE-antigen complexes,neither miR-221 overexpression, nor its ablation, interfered with mast cell differentiation. Transcriptional profiling of miR-221-overexpressing mast cells revealed modulation of many transcripts, including several associated with the cytoskeleton;indeed, miR-221 overexpression was associated with reproducible increases in cortical actin in mast cells, and with alteredcellular shape and cell cycle in murine fibroblasts. Our bioinformatics analysis showed that this effect was likely mediated bythe composite effect of miR-221 on many primary and secondary targets in resting cells. Indeed, miR-221-induced cellularalterations could not be recapitulated by knockdown of one of the major targets of miR-221. We propose a model in whichmiR-221 has two different roles in mast cells: in resting cells, basal levels of miR-221 contribute to the regulation of the cellcycle and cytoskeleton, a general mechanism probably common to other miR-221-expressing cell types, such as fibroblasts.Vice versa, upon induction in response to mast cell stimulation, miR-221 effects are mast cell-specific and activation-dependent, contributing to the regulation of degranulation, cytokine production and cell adherence. Our studies providenew insights into the roles of miR-221 in mast cell biology, and identify novel mechanisms that may contribute to mast cell-related pathological conditions, such as asthma, allergy and mastocytosis.

Citation: Mayoral RJ, Deho L, Rusca N, Bartonicek N, Saini HK, et al. (2011) MiR-221 Influences Effector Functions and Actin Cytoskeleton in Mast Cells. PLoSONE 6(10): e26133. doi:10.1371/journal.pone.0026133

Editor: Jose Alberola-Ila, Oklahoma Medical Research Foundation, United States of America

Received May 10, 2011; Accepted September 20, 2011; Published October 12, 2011

Copyright: � 2011 Mayoral et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: RJM was the recipient of a pre-doctoral fellowship from the San Raffaele University (Italy). This work was supported by the Swiss National ScienceFoundation grant 31003A_121991 to SM and by a contribution from the Ceresio Foundation (Switzerland) to SM. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

Mast cells are cells of the innate immune system that reside in

most tissues and are characterized by cytoplasmic granules

containing active mediators such as histamine and proteases,

which can be rapidly released upon activation. A broad panel of

cytokines and chemokines is also rapidly synthesized upon acute

stimulation, although some cytokines (such as TNFa) can be pre-

stored in the granules [1,2]. On the surface, mast cells are

characterized by the expression of KIT and FceRI (high-affinity

IgE receptor). KIT, the receptor for the stem cell factor (SCF), is a

critical regulator of mast cell differentiation, proliferation,

activation and survival. Although mast cells have been considered

for a long time detrimental to the host, mainly because of their

major role as master effector cells in allergic responses, it is now

becoming clear that depending on the context, mast cells can

either positively or negatively regulate innate or adaptive immune

responses to pathogens or allergens (for a recent review see [3]).

MicroRNAs (miRNAs) are small, non-coding RNAs that are

emerging as major players in the regulation of endogenous gene

expression in immune homeostasis [4]. Specifically, miRNAs

regulate the expression of target genes by interacting with sites in

the 39 untranslated region (39UTR) of their target mRNAs.

MiRNAs regulate all aspects of a cell, including differentiation,

function, proliferation, survival, metabolism, and responses to

environmental changes. A single miRNA can potentially modulate

the expression of hundreds of transcripts in a cell, both by direct

and indirect effects, and as many as 90% of human genes may be

regulated by miRNAs [5]. MiRNAs are implicated in various

pathologic conditions as well as in tumorigenesis [4,6], and within

the immune system, deletion or overexpression of specific miRNAs

can impair innate or adaptive immune responses [7].

While the importance of miRNAs in various types of normal

and diseased cellular processes is by now well established, very

little is known about the role of miRNAs in mast cell development,

function and disease. Our lab identified miR-221/-222 as a family

of miRNAs that is transcriptionally induced upon mast cell

activation, and we showed that expression of miR-221 and/or

miR-222 to levels similar to the endogenous of activated mast cells,

led to reduced mast cell proliferation [8]. MiR-221 and miR-222

PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26133

derive from the same primary transcript and share the same seed

sequence, implying that they should recognize the same targets

[9]. Following up on our previous studies, we investigated the role

of miR-221 in mast cell differentiation and function. Specifically,

we found that although miR-221 does not seem to affect mast cell

differentiation, it has important roles in regulating multiple

processes in differentiated mast cells, such as degranulation,

adhesion and migration, some of which may be linked to a

dysregulation in the actin cytoskeleton. Indeed, we found that

alteration of miR-221 expression in mast cells and fibroblasts led

not only to a reduction in cell proliferation similar to what we

previously described using a different expression system, but also

to an alteration of actin content and overall cellular shape in both

cell types. Transcriptional profiling and bioinformatics analysis

using the Sylamer algorithm [10], indicated that miR-221 effects

in mast cells were mediated by the alterations of the level of

expression of many primary and secondary targets. Importantly,

such miR-221-mediated alterations of the cell phenotype could not

be recapitulated by knockdown of one of the most prominent

target for miR-221, suggesting that the observed effect of miR-221

in resting mast cells and fibroblasts is likely to be composite, due to

the alteration of many genes. Moreover, we also observed mast

cell-specific, activation-dependent effects of miR-221. Since miR-

221 is expressed at basal level in mast cells, but it is also inducible

upon stimulation, we propose a model in which miR-221 has a

dual roles in these cells: at resting state, it contributes to the

regulation of the cell cycle and cytoskeleton, a housekeeping effect

that can be observed also in different cell types expressing this

miRNA. However, in response to stimulation through IgE-antigen

complexes, miR-221 effects are mast cell-specific and activation-

dependent, contributing to the regulation of degranulation,

cytokine production and cell adherence. Overall, our studies

provide insights on the role of miRNAs in mast cells, and lay the

groundwork for understanding some of the mechanisms underly-

ing pathological conditions caused by mast cells, such as allergy

and mastocytosis.

Materials and Methods

Ethics statementAll animal studies were performed in accordance with the Swiss

Federal Veterinary Office guidelines and were approved by the

Dipartimento della Sanita’ e della Socialita’, authorization number

18/2010.

PlasmidsAbout 400bp of the mouse miR-221 or miR-222 genomic

sequences were cloned into the pAPM lentiviral vector [11]. Point

mutations in the seed sequence of miR-221 were introduced using

the Quick Change Site-Directed Mutagenesis kit (Stratagene) and

the following primers (mutations underlined): miR-221mFW: 59-

GTTTGTTAGGCAACATCGCGATTGTCTGCTGGGTTTC-

AGG; miR-221mRV: 59- CCTGAAACCCAGCAGACAATCGC-

GATGTTGCCTAACAAAC. The control vector pAPM-shLuc,

expressing a miR-30-based shRNA against luciferase, was obtained

from Thomas Pertel and Jeremy Luban and contained the following

sequence: 59- CACAAACGCTCTCATCGACAAG. The miRNA

target (miRT) vectors containing four sequences fully complemen-

tary to miR-221 and/or miR-222, were provided by Bernhard

Gentner and Luigi Naldini [12].

Cell culturesFor bone marrow-derived mast cells (BMMCs) differentiation,

bone marrow cells from C57Bl/6 mice (6-8 weeks old) were

differentiated and maintained in IMDM with 10% FBS, 2 mM L-

glutamine, 0.1 mM non essential amino acids, 50 mMb-mercap-

toethanol, antibiotics and 50% WEHI-3 conditioned supernatant

as a source of IL-3 [13,14]. The IL-3-dependent mast cell line

MC/9 (ATCC) was cultured as for primary BMMCs. 3T3 cells

were cultured in DMEM supplemented with 10% FBS, 2 mM L-

glutamine, 0.1 mM non-essential amino acids, 50 mMb-mercap-

toethanol and antibiotics.

Transductions and transfectionsLentiviral transductions of mast cells were performed exactly as

described [8,13]. 3T3 cells were transduced with concentrated

lentiviruses prepared exactly as described [8,13] and selected with

2 mg/mL puromycin for several days before performing experi-

ments. For transient transfections with oligonucleotides, 3T3 cells

were transfected with Lipofectamine-2000 (Invitrogen) and 20pmol

of siRNAs against p27Kip1 (siGENOME Cdkn1b) or control siRNAs

(siGENOME non-targeting siRNA #2 and/or siGLO) (all from

Thermo Scientific) following manufacturer’s instructions. Cells were

analyzed 48–72 h after transfection. Efficiency of transfection was

assessed in each experiment by transfecting a fluorescent oligonu-

cleotide (siGLO) and analyzing the percentage of siGLO+ cells by

FACS, which usually ranged between 50 and 70%. The efficiency of

protein knockdown was assessed by Western blot.

RNA extraction and RT-PCRTotal RNA was extracted using TRIzol (Invitrogen) according

to manufacturer’s instructions. To analyze miRNA expression,

qRT-PCR was performed using a miRNA reverse transcription kit

and TaqMan miRNA assays from Applied Biosystems, following

exactly manufacturer’s instructions.

Immunostainings and microscopyAnti-KIT-APC, anti-FceRIa-PE and anti-CD25-PE were

purchased from eBioscience. For microscopy, 105 BMMCs were

labeled with CFSE (Invitrogen) and added to a monolayer of 3T3

cells on coverslips. After 12 hours, coverslips were washed to

eliminate non-adherent cells, and cells were fixed with 3.7% p-

formaldehyde, permeabilized with 0.1% Triton-X100 and stained

with 160nM phalloidin-AlexaFluor-594 (Invitrogen). Glass slides

were mounted using Gelvatol (20% polyvinyl alcohol, 100mM

Tris-HCl pH 8.5, 2.5% DABCO). The same protocol was used to

stain 3T3 cells grown directly on coverslips. Bright field images

were captured with a Nikon Eclipse E800 and analyzed with the

Openlab software (Improvision). For FACS-based quantification

of F-Actin, 3?105 cells were fixed 15–30 min in 4–5% p-

formaldehyde, permeabilized 2min with 0.1% Triton-X100, and

stained with phalloidin at a 1:100 dilution.

Degranulation, adhesion and migration assaysFor degranulation assay, BMMCs (5?104) were resuspended in

50 mL OptiMEM, 1% FBS and stimulated for 1 h with 1.5 mg/mL

IgE-anti-DNP (clone SPE-7, Sigma) and 0.2 mg/mL DNP-HSA

(Sigma). After stimulation, cell pellets were lysed in 50 mL of 0.5%

Triton-X100 in OptiMEM, 1% FBS, and 50 mL of a 3.8 mM

solution of the b-hexosaminidase substrate 4-nitrophenyl N-acetyl-

b-D-glucosaminide (Sigma) were added to both cell lysates and

supernatants. After incubation for 2 h at 37uC, the reaction was

stopped with 90 mL glycine 0.2 M, pH 10.7, and absorbance was

read at 405 nm. The percentage of degranulation was calculated as

the ratio between the absorbance of supernatants and the total

absorbance of supernatants and cell lysates [15]. Alternatively,

degranulation was assessed using the same annexin V-PE kit used to

Effects of miR-221 in Murine Mast Cells


detect apoptosis (BD-Pharmingen) [16,17]. Briefly, cells were

stimulated for 30 min with 1.5 mg/mL IgE-anti-DNP and 0.2 mg/

mL DNP-HSA, in the presence or absence of 20 ng/mL SCF

(Peprotech). Cells were then washed and stained with annexin V-PE

following manufacturer’s instructions. For adhesion experiments,

105 BMMCs were added to a monolayer of 3T3 cells in 24-well

plates. Floating cells were harvested 8h later, and adherent cells

were detached using 10mM EDTA. In order to distinguish BMMCs

from fibroblasts, cells were stained for KIT, and numbers of KIT+cells were evaluated by FACS. The percentage of adherent cells was

calculated as the ratio between adherent cells versus total. Cell

migration was assayed using 24-well transwell chambers (Corning)

with 8.0 mm pores in polycarbonate membranes. 2?105 BMMCs

were seeded in the upper chambers and allowed to migrate for 2 h.

In some experiments, 20 ng/mL of SCF was used as chemoat-

tractant, while in others BMMCs were first sensitized with 300 ng/

mL IgE-anti-DNP for 12 h and then allowed to migrate towards

0.2 mg/mL DNP-HSA. The percentage of migrated cells was

calculated as the ratio between the number of cells in the lower

chamber versus total.

Cell-cycle analysis and Western blotsFor propidium iodide staining and DNA content analysis, non-

confluent 3T3 cells were fixed in 70% ethanol for 45 min on ice,

followed by incubation for 30 min at 37uC with 100 mg/mL

RNaseA and 40 mg/ml propidium iodide. Cells were analyzed by

FACS immediately afterwards. Total protein extracts for Western

blot were prepared by cell lysis in Laemmli sample buffer. Samples

were run on 12% SDS-polyacrylamide gels and immunodetection

was performed with p27 C-19 and b-tubulin H-235 antibodies

(Santa Cruz Biotechnologies).

Microarrays and Sylamer analysisGene arrays were performed at Miltenyi Biotec using Agilent

dual-color whole-genome oligo arrays and total RNA from three

independent biological replicas. Genes found to be commonly

regulated were functionally annotated. Sylamer analysis was

performed through a Sylarray web server [18,19]. Specifically, the

Agilent probes were mapped to the 39zUTRs of genes stored in the

Sylarray database. The sequences were previously masked from low

complexity regions and redundant UTR sequences with DUST

algorithm and RSAT purge-sequence interface to Vmatch [20,21].

As a control we also analyzed the mutant seed sequence to check for

any evidence of its influence in the experiment and no significant

association was found. Array data are MIAME compliant and are

deposited in the MIAME compliant database Gene Expression

Omnibus (GEO) with accession number GSE24462.

Statistical analysisResults are expressed as mean 6 standard deviation. Compar-

isons were made using the Student’s unpaired t-test and the

GraphPad Prism Software.

Supporting InformationAdditional information can be found in the Supplementary

Methods S1.

Results

A robust lentiviral system for manipulating microRNAexpression in mast cells

As we previously described for acute stimulation of mast cells

[8], BMMCs stimulated with IgE-antigen complexes upregulated

miR-221 expression (Figure 1A). While stimulation-dependent

upregulation of this miRNA could be favored by SCF co-

stimulation, SCF alone had no effect on miR-221 expression (not

shown). To investigate the role of miR-221 in regulating primary

mast cell functions, we developed a lentiviral system to manipulate

miRNA expression in primary BMMC and used it to alter miR-

221 expression. The pAPM/pAGM vectors were used to

overexpress miR-221 or miR-222; as control, we used a mutant

version of miR-221 (miR-221m), containing mutations in the seed

region to abrogate target recognition, as well as a vector expressing

an irrelevant hairpin (shLuc) (Figure 1B). The miR-221m mature

sequence had no predicted targets as assessed by TargetScan [9].

The ‘miRNA target’ (miRT) vectors contain four miRNA binding

sites (miR-bs) cloned downstream a GFP reporter gene, and they

were used to functionally ablate miR-221/-222 [12]. Transcrip-

tion from such vectors results in accumulation of decoy mRNAs

that divert miRNAs from their physiological targets [22].

To assess expression from these vectors, BMMCs were

transduced with the indicated vectors, and miRNA expression

was assessed by qRT-PCR (Figures 1C and 1D). Compared to

untransduced, unstimulated cells (expression set to 1 in Figures 1C

and 1D), transduction of primary mast cells with pAGM/pAPM-

miR-221 increased miR-221 expression by ,60-fold, whereas

transduction with miRT-221 decreased expression by ,10-fold

(Figure 1D). Transduction with the mutant miR-221m had no

effect (Figure 1C). Initial experiments were performed using a

vector (Tween) that induced only modest overexpression (,4-fold),

similar to the levels of endogenous miR-221 observed upon cell

stimulation (Figure 1A) [8]. However, both types of vectors

(weaker and stronger expression) gave similar results qualitatively,

although the stronger vector provided bigger quantitative

differences, and was therefore used in most of the subsequent

experiments. To assess the functional effects of miRNA overex-

pression/ablation, the mast cell line MC/9 was transduced to

overexpress miR-221 or the mutant miR-221m. Transduced cells

were selected with puromycin, subjected to a second round of

transduction with the miRT vectors, and monitored for GFP

expression (Figure 1E). As a result of binding of the overexpressed

miRNAs to their cognate sites in the 39 UTR of the GFP reporter

mRNA expressed from the miRT, GFP expression was strongly

reduced specifically in cells expressing miR-221 but not the

mutant miR-221m. We therefore used both validated systems

(overexpression and ablation) to study mast cell differentiation in

the presence or absence of miR-221.

MiR-221/-222 as well as the transcriptional repressor PLZF are

both known important regulators of hematopoietic cell differen-

tiation [23,24,25]. We previously showed that binding sites for

PLZF were enriched in mast cell-specific DNaseI hypersensitive

sites found upstream of the miR-221-222 genomic sequence [8].

To address the possible relation between PLZF and miR-221, we

analyzed expression of both Plzf mRNA and miR-221 during mast

cell differentiation (Supplementary Figure S1). We observed an

inverse relation between Plzf and miR-221 expression during mast

cell differentiation, and ectopic expression of PLZF in mast cells

diminished miR-221 expression in response to acute stimulation,

suggesting that PLZF is able to repress miR-221-222 induction

either directly or indirectly, and possibly through PLZF-binding

regulatory elements in the miR-221-222 locus [8]. However,

ectopic expression of PLZF in differentiated mast cells had no

effect on the basal levels of endogenous miR-221, indicating that

other factors regulate basal expression of this miRNA in mast cells.

To assess whether miR-221/-222 may have a direct role in

regulating the differentiation process in mast cells, we transduced

bone marrow-derived hematopoietic progenitors with lentiviruses



to either overexpress (pAPM) or ablate (miRT) miR-221 and/or

miR-222 early during mast cell differentiation (Supplementary

Figure S1). Differentiation was monitored over a period of at least

three weeks by assessing the percentage of FceRIa+ KIT+ cells.

Interestingly, the percentage of BMMCs increased steadily over

time in all samples, and mast cell differentiation was not

significantly affected by either overexpression or ablation of

miRNAs. Moreover, there was no obvious alteration in cell

granularity or in the content of the granules (data not shown).

MiR-221 regulates degranulation, migration andadherence in differentiated BMMCs

Since there was no effect of miR-221 in mast cell differentiation,

we set out to investigate its role in mast cell functions, especially

the ones connected to signaling through the FceRI, given that

miR-221 expression is inducible upon stimulation. Differentiated

BMMCs were lentivirally transduced to force expression of miR-

221, followed by analysis of the effects on mast cell degranulation,

migration and adherence (Figure 2). Upon activation, mast cells

release an array of enzymes that are pre-stored in cytoplasmic

granules. We analyzed the ability of miR-overexpressing BMMCs

to degranulate upon stimulation using b-hexosaminidase activity

in the supernatant of activated cells as a measure of degranulation

(Figure 2A). In resting conditions, cells did not degranulate,

regardless of miRNA expression (Figure 2A, top panel) but, upon

stimulation with IgE and antigen, BMMCs overexpressing miR-

221 degranulated more compared to the controls (Figure 2A, lower

panel), although they also showed a slightly reduced content of b-

hexosaminidase in the granules to begin with (Figure 2A, top panel).

To further confirm these results and to assess the effect of different

conditions of stimulation (namely IgE crosslinking with or without

SCF co-stimulation), we assessed degranulation of cells overex-

pressing miR-221 or controls by using a staining with annexin V.

This staining takes advantage of the fact that mast cells do not die

upon stimulation (which is instead a survival factor [26]) and that

during the membrane fusion process of degranulation, annexin V

binding occurs at sites of secretory granule exposure to the cell

surface [16,17]. In general, BMMCs stimulated with a combina-

tion of IgE-antigen and SCF degranulated more strongly

compared to cells stimulated without SCF (Figure 2B). However,

Figure 1. MiR-221 is upregulated upon mast cell activation and its expression levels can be altered using lentivirus-based systems.A) Differentiated BMMCs were either left resting or were stimulated with 1.5 mg/mL IgE anti-DNP, 0.2 mg/mL DNP-HSA and 20ng/mL SCF prior analysis ofmiR-221 expression by TaqMan qRT-PCR. SnoRNA202 was used as endogenous control. B) Schematic representation of the lentiviral vectors used tostably overexpress or functionally ablate miR-221 and miR-222 in BMMCs. Reporter genes were either puromycin or ZsGreen in the pAPM or pAGMvectors, respectively. Sequences corresponding to the mature murine miR-221, miR-222 and miR-221m are also shown. The miRT vectors contain 4sequences fully complementary to miR-221 and/or miR-222 cloned downstream the reporter gene. SFFVp: spleen focus-forming virus promoter;d4EGFP: destabilized GFP; bs: binding sites. C) BMMCs were transduced with the control vector shLuc, miR-221 or miR-221-mutant. After selection with2 mg/mL puromycin for 48h, miR-221 expression levels were assessed by TaqMan qRT-PCR. Expression levels are referred to the ones of the shLuc-transduced cells, which are set to one. D) BMMCs were transduced with miRT-221 (depleting) or miR-221 (overexpressing) vectors, and miR-221expression levels were assessed by TaqMan qRT-PCR. Cells transduced with miR-221 were selected with 2 mg/mL puromycin for 48h prior analysis, whilecells transduced with miRT-221 were sorted to .90% GFP+ prior RNA extraction. Untransduced cells were either left unstimulated or were stimulatedwith PMA and ionomycin for 24 h prior RNA extraction. Expression levels are referred to the untransduced and unstimulated cells, which are set to one.E) MC/9 cells were transduced with miR-221 (upper panels) or miR-221m (lower panels) and selected with 1 mg/mL puromycin prior a secondtransduction with the indicated miRT vectors (miRT-empty or miRT-221). GFP levels were determined by FACS. Transduction efficiency and levels of miR-221 expression or depletion are representative of tens of experiments, including all the experiments shown hereafter.doi:10.1371/journal.pone.0026133.g001



compared to the controls (shLuc and miR-221m) miR-221

overexpression increased degranulation in response to IgE, as

shown already by b-hexosaminidase assay, but miR-221-overex-

pressing cells did not further degranulate in response to the

combination of both SCF and IgE crosslinking (middle panel).

This could be due to the fact that the SCF receptor KIT is

expressed at lower levels on these cells (see below), or to the fact

that in the presence of miR-221 cells are activated more strongly

upon IgE crosslinking, and cannot be further activated by the

combination of IgE and SCF.

Next, we investigated the capability of BMMCs to migrate in a

transwell system (Figure 2C). We found that cells overexpressing

miR-221 migrated significantly less towards SCF as compared to

the controls (Figure 2C, right panel). KIT is a target for miR-221

[24], therefore, to understand whether the reduced migration was

due to an intrinsic feature of cells overexpressing miR-221, or to a

reduced ability to ‘sense’ SCF in the environment due to lowered

expression of KIT, we repeated the same experiment sensitizing

BMMCs with IgE-anti-DNP prior inducing migration towards

DNP-HSA (Figure 2C, left panel). The discernible, albeit modest,

migration of control-transduced cells towards the antigen was

significantly impaired if miR-221 was overexpressed, indicating

that the reduced migration was due to effects of miR-221 on

targets other than KIT.

Another process promoted by the stimulation through the

FceRI is adherence of mast cells to the substrate. Since mature

mast cells do not normally circulate in vivo, but reside in tissues, we

explored whether miR-221 had any role in regulating cell

adherence and migration, as these are essential processes not only

under normal homeostatic conditions, but also during inflamma-

tion and tumorigenesis. As in vitro-differentiated mast cells grow in

suspension, we assessed the ability of BMMCs to adhere to a

feeder layer of fibroblasts in a co-culture system (Figure 2D).

Increased adherence is a normal process observed upon

stimulation of mast cells with IgE and antigen, however, BMMCs

expressing miR-221 adhered at a higher percentage compared to

the controls even in resting, unstimulated conditions. Vice versa,

upon stimulation all cells were able to adhere to the feeder layer of

fibroblasts at comparable levels, regardless of miRNA expression.

Since miR-221 overexpression was sufficient by itself to increase

adherence, our data indicate that endogenous miR-221/-222

upregulation upon cell activation may contribute to the increased

adherence of mast cells observed upon stimulation.

These results may point towards a role for miR-221 in

regulating the signaling cascade originating from the FceRI. In

this context, it is important to highlight that miR-221 overexpres-

sion did not alter surface expression of FceRI (see below). To

evaluate whether signaling from the FceRI could be affected in

presence of miR-221, we assessed the levels of ERK phosphor-

ylation in response to IgE crosslinking (Supplementary Figure S2).

In all conditions and time-points tested, there was no significant

difference in ERK phosphorylation in cells overexpressing miR-

Figure 2. Degranulation, migration and adherence of cells overexpressing miR-221. A) Transduced BMMCs were either left unstimulated(upper panel; n = 5 experiments, each performed in triplicate) or were stimulated with IgE anti-DNP and DNP-HSA for 1h (lower panel; n = 2experiments, each performed in triplicate) prior measurement of b-N-acetylhexosaminidase release to assess degranulation. B) Transduced BMMCswere either left unstimulated or were stimulated for 30min with IgE-antigen complexes alone or in combination with 20ng/mL of SCF. Cells were thenstained with annexin V-PE to assess the extent of degranulation (representative of 2 independent experiments). The red shading in the figureindicates the difference between IgE versus IgE+SCF stimulation in control cell, which is reduced in miR-221-expressing cells). C) Right panel: BMMCstransduced with indicated vectors were seeded in the upper well of a transwell chamber and then allowed to migrate towards the lower chambercontaining no chemoattractant (empty circles) or SCF (black circles). Each circle represents one independent experiment. Left panel: same as above,except that cells were sensitized with anti-DNP IgE overnight, and allowed to migrate towards the antigen (DNP-HSA). D) BMMCs transduced with theindicated vectors were seeded on a monolayer of 3T3 cells, and were either left resting or were stimulated for 8h with IgE-antigen complexes andSCF. Cells were then detached by treatment with 10mM EDTA, and the percentage of adherent cells versus total was assessed by surface staining forKIT and FACS analysis. Shown is one experiment out of at least six.doi:10.1371/journal.pone.0026133.g002



221 compared to the controls, suggesting that miR-221 might not

affect directly the signaling cascade from the FceRI. However, in

miR-221-expressing cells, in addition to increased degranulation

(Figure 2, panels A and B), we also observed increased cytokine

production (IL-6 and TNFa) in response to IgE crosslinking, but

not to LPS (Supplementary Figure S2 and data not shown). This

observation may indicate that miR-221 expression favors mast cell

activation in response to IgE-antigen complexes, however in a way

that doesn’t seem to grossly affect ERK phosphorylation.

Microarray analysis identified genes affected by miR-221To gain insight into the mechanisms underlying such pleiotropic

effects of miR-221 in mast cells, we performed a microarray

analysis of BMMCs overexpressing miR-221 or miR-221m, as it

has been reported that indeed the impact of a miRNA on protein

production can be closely approximated using mRNA arrays [27].

Out of ,42.000 transcripts analyzed, we found 397 significantly

upregulated genes in miR-221-expressing BMMCs, as well as 343

significantly downregulated genes as compared to cells transduced

with miR-221m (Figure 3A). Importantly, the known miR-221

targets Cdkn1b (p27Kip1) and Kit were found to be downregulated

with a mean fold-change repression of -3.2 and -2.0, respectively.

While the upregulated genes presumably reflect secondary

changes due to changes in the expression of primary targets, the

downregulated gene subset may contain both primary targets as

well as secondary effects.

Of note, when we compared the list of our downregulated genes

with the targets predicted by TargetScan [9] and microCosm [28],

we found very few overlapping genes, which could indicate either

that only few downregulated genes are the real primary targets, or

that many targets are relevant only in the specific mast cell context

(Figure 3B). To better understand how miR-221 could regulate

gene expression specifically in mast cells, we therefore performed a

Sylamer analysis [19] on the complete gene list from the arrays,

which was ranked from most downregulated to most upregulated

(left to right in the graph in Figure 3C). Sylamer is an algorithm for

determining whether specific 6, 7 or 8nt motifs corresponding to

miRNA seed sequences are enriched or depleted in a ranked gene

list [19]. The Sylamer plots can be used to analyze influence of a

miRNA on an expression set profile. Sylamer calculates enrich-

ment of seed words in 39UTRs of a sorted gene list, providing a p-

value for genes left of each rank versus the genes on the right. In

this case, the upper left portion of the plot represents an influence

of miRNAs on downregulated genes (Figure 3C), indicating a

specific influence of miR-221 on a subset of downregulated genes.

Any point of a line provides a p-value that a word is enriched in

the UTRs left of it on the X-axis. Although in some cases most of

the effect of a given miRNA in a particular cellular context goes

Figure 3. Identification of genes dysregulated upon miR-221 overexpression in BMMCs. A) Double-log scatter plot comparing thedifferential expression of mRNAs in BMMCs transduced with the miR-221 and miR-221m. Red diagonal lines define the areas of 2-fold differentialsignal intensities. Blue cross: unchanged genes. Red cross: significantly upregulated genes (p-value ,0.01). Green cross: significantly downregulatedgenes (p-value ,0.01). B) Comparison between significantly downregulated genes and genes predicted to be miR-221 targets by microCosm andTargetscan. C) Sylamer plot analysis for the Seed Complementary Region (SCR) words corresponding to the seed of miR-221 (red) and its one-nucleotide shifted sequence (orange). Log10- transformed and sign-adjusted enrichment P values for each SCR word, relative to P values of all otherwords, are plotted on the Y-axis, against the ranked gene list on the X-axis (left, downregulated genes; right, upregulated genes). The dashed greenline defines the Bonferroni corrected p-value cutoff for significance of 0.05.doi:10.1371/journal.pone.0026133.g003



primarily through one or few molecular targets, in other cases this

does not provide the complete picture of miRNA effect on the

transcriptome and proteome of a cell [27,29,30]. Our array

analysis showed that the transcriptomes of miR-221-overexpress-

ing cells differed by the expression of 740 genes compared to the

controls, and the Sylamer analysis indicated that many of the

downregulated genes in mast cells were likely to be directly

affected by miR-221 expression, suggesting that the effect of this

miRNA goes through the fine modulation of a multitude of targets.

Some of the targets that were up- or downregulated were also

confirmed at protein level; among others, we selected two of the

already known targets for miR-221, Kit and Cdkn1b [8,24,31], as

well as one upregulated gene (Il2Ra) that is particularly relevant in

mast cell biology as a marker for systemic mastocytosis [32,33]. As

assessed by surface staining, levels of KIT, but not of FceRI, were

significantly diminished (,2-fold) in BMMCs overexpressing miR-

221 (Figure 4A). In contrast, as we previously described [8], levels

of p27Kip1 protein were especially decreased in stimulated cells

(Figure 4B, compare lane 4 with lanes 2 and 6). While CD25

(Il2Ra) is normally not expressed by human mast cells in non-

pathogenic conditions [32,33], it is normally expressed by a subset

of in vitro-differentiated murine BMMCs (Figure 4C) as well as by a

subset of peritoneal and tissue mast cells in the mouse (Deho’ and

Monticelli, unpublished observation). The mean fold increase for

Il2Ra from the arrays was 2.1, which strongly correlated with the

increased surface expression of this marker in cells overexpressing

miR-221 (Figure 4C).

Although some of the targets for miR-221 were individually

confirmed, our arrays experiments and Sylamer analysis indicated

that miR-221 determines the downregulation of more than 200

primary targets and the subsequent secondary alterations of many

genes in the transcriptome. Therefore, to gain insights into the

function of the genes that showed altered expression in the presence

of miR-221, we performed functional grouping and gene ontology

Figure 4. MiR-221 primary and secondary targets are altered also at protein level. A) Differentiated BMMCs were either left untransducedor were transduced with the indicated vectors. After selection with puromycin, surface expression of KIT (left) and FceRI (right) was assessed by FACS.The mean fluorescence intensity (MFI) for KIT expression in miR-221- (red) and shLuc- (black) expressing cells is also indicated. B) DifferentiatedBMMCs as in A) were either left untreated or were stimulated with 20nM PMA and 1 mM ionomycin for 24h prior lysis and Western blot analysis ofp27Kip1 expression. b-tubulin was used as loading control. C) BMMCs as in A) were stained and FACS-analyzed to assess surface expression of KIT andCD25. The horizontal dotted line was added as a reference to appreciate the expected KIT dowregulation in miR-221 expressing cells.doi:10.1371/journal.pone.0026133.g004



(GO) analysis, and found that many basic biological processes were

affected (Table 1). Among the downregulated genes, the categories

that were statistically significant were surprisingly few, and

belonging for the most part to very general metabolic pathways,

such as protein folding, transcription, lipid metabolism and cell

differentiation. This finding may suggest that in resting, unstimu-

lated cells, miR-221/-222 regulate basic metabolic processes that

are common to many different cell types and/or species, and

correlates with the fact that these miRNAs belong to a family very

conserved in evolution, down to at least zebrafish [8]. Although they

are most likely due to secondary changes in the transcriptome, the

GO categories of the upregulated genes included several pathways

that we also observed altered experimentally, such as cell

proliferation, cell adhesion and cell migration. Among these latter

GO categories, we focused our attention on one of the most

statistically significant one, namely the cytoskeleton (which was also

present in the GO list of terms for the downregulated genes,

although with a p-value of 0.1), as we hypothesized that an

alteration in the ability of the cells to properly regulate the

cytoskeleton could to some extent explain why these miRNAs affect

many different biological processes, at least in unstimulated cells.

MiR-221-dependent alterations of the cytoskeleton andactin content in mast cells and 3T3 cells

To assess whether miR-221 expression could affect the

cytoskeleton, we stained transduced BMMCs with phalloidin

(Figure 5A). We used the same co-culture system with fibroblasts

used in Figure 2D to assess cell adherence, and to distinguish mast

cells from the underlying layer of feeder cells (also stained by

phalloidin), BMMCs were first labeled with CFSE. Strikingly, not

only miR-221-overexpressing cells showed increased numbers of

adherent cells (as shown also in Figure 2D), but while the actin ring

underneath the plasma membrane was barely visible in control

cells, cells overexpressing miR-221 (or miR-222, not shown)

showed the presence of a much thicker ring (Figure 5A).

Moreover, when we quantified the overall cellular amount of F-

actin in cells depleted for miR-221 (using the miRT-depleting

vectors), we observed a small but reproducible decrease in the

amount of F-actin present in these cells (Figure 5B), further

indicating that these miRNAs might be important regulators of the

actin organization in mast cells.

To independently confirm these results, and to investigate

whether the observed effect was a general feature of this miRNA

or a cell type-specific effect due to alterations of targets relevant

only in the mast cell context, we transduced 3T3 fibroblasts with

the same lentiviral vectors used on mast cells. 3T3 cells expressed

low levels of endogenous miR-221 that were increased ,20-folds

upon transduction with a miR-221 expressing vector (Figure 6A).

MiR-221 overexpression in 3T3 cells led to a strong downregu-

lation of endogenous p27Kip1, even more remarkable than the one

observed in mast cells (Figure 6B). Despite such strong

downregulation of the cell-cycle inhibitor p27Kip1, 3T3 cells

overexpressing miR-221 showed the same reduced proliferation

that we previously described for mast cells (Figure 6C) [8].

Moreover, 3T3 cells overexpressing miR-221 showed overall

altered morphology, with odd, elongated and/or irregular shapes

(Figure 6D), as well as a slightly increased content of F-actin

(Figure 6E), indicating that the miR-221-dependent effects on the

cytoskeleton and cell cycle observed in resting mast cells are likely

to be due to the dysregulation of targets that are ubiquitously

expressed and are therefore cell type-independent. However,

FceRI stimulation led to mast cell-specific (or at least not present in

fibroblasts) effects of miR-221, with increased degranulation and

cytokine production.

Analyzing the data from our transcriptome profiling, we found

that in the ‘cytoskeleton’ group of downregulated genes, the top

candidate, most downregulated gene was Cdkn1b (p27Kip1), and

specifically the one splice variant that can be regulated by miR-

221/-222 (as we previously described in [8]). While p27Kip1 is a

cell cycle inhibitor with a well established role in cell cycle

progression at the G1-S transition, it has also been shown that

cytoplasmic p27Kip1 plays an important role in cell motility and

migration, and that p27Kip1-deficient fibroblasts fail to form long

cellular protrusions, assume an overall rounded shape [34] and

show reduced migration [35]. To assess whether miR-221-

dependent down-regulation of p27Kip1 may have a role in

regulating 3T3 and mast cells shape and cytoskeleton, we therefore

performed a knockdown of p27Kip1 in 3T3 cells using siRNAs

(Figure 7). Efficiency of transfection and p27Kip1 knockdown were

evaluated by transfection and FACS analysis of a fluorescent ds-

oligo (siGLO) and by Western blot, respectively (data not shown

and Figure 7A). It has to be noted that the efficiency of transfection

was at the most ,70%, so that the residual protein observed in

Western blot may in part be due to the fact that some cells still

expressed significant levels of p27Kip1. However, the knockdown of

p27Kip1 did not alter the overall cell-cycle profile of 3T3 cells

(Figure 7B), and the cells did not show any particularly altered

shape, apart from a slight increase in the percentage of cells that

were smaller and more rounded (Figure 7, panels C and D).

Although this effect was fairly modest (even in the experiments

Table 1. Gene Ontology (GO) frequency distribution fordownregulated and upregulated genes.

DOWNREGULATED –Categories Times Observed Probability

Cell migration 15 5.30E-05

Protein folding/modification 30 1.80E-04

Transcription 38 5.70E-04

Lipid metabolism 17 2.30E-03

Cell differentiation 28 3.20E-03

Other metabolism 29 8.00E-03

UPREGULATED – Categories Times Observed Probability

Receptor signaling 73 9.40E-09

Cell proliferation 26 4.60E-08

Cytoskeleton 35 4.70E-08

Cell cycle 27 6.40E-08

Cell adhesion 26 3.30E-07

Cell differentiation 37 1.10E-05

Kinase/Phosphatase signaling 23 1.30E-05

Other metabolism 39 1.90E-05

Protein folding/modification 35 2.50E-05

Development 46 2.90E-05

G-protein signaling 43 8.80E-05

Cell migration 15 3.10E-04

Nucleotide metabolism 26 3.70E-04

Inflammation 11 1.00E-03

Intracellular trafficking 20 5.08E-03

Gene ontology (Bonferroni corrected) of mRNAs that were downregulated orupregulated in the microarrays. A cutoff of P = 0.01 was used for the categoriesto be considered statistically significant.doi:10.1371/journal.pone.0026133.t001



with the strongest downregulation of p27Kip1 only ,15% of the

cells were counted as ‘small and round’, compared to the controls),

it was in line with what was previously reported for Cdkn1b-deleted

fibroblasts. Indeed, p27Kip1 KO fibroblasts were shown to have a

rounded shape with no alterations in the cell cycle [34]. Most

importantly, the knockdown of p27Kip1 did not recapitulate the

phenotype we observed in miR-221 overexpressing 3T3 cells, as

cell cycle and cellular shape were either unaltered or completely

different from what we observed in miR-221-transduced cells,

suggesting (as indeed indicated by our Sylamer analysis), that the

effect of this miRNA is composite and goes through the down-

modulation of multiple targets.

Although the mechanisms underlying the role of miR-221

specifically in mast cells in both resting and stimulated conditions

will require further investigation and will be the subject of future

work, our data show that the effect of this miRNA goes through

the alteration of the levels of many targets in the mast cell

transcriptome, that it has important roles in regulating mast cell

physiology, and finally that at least some of its biologic effects in

resting cells may be explained by alterations in the actin

cytoskeleton of mast cells.

Discussion

Although mast cells have a long lifespan, accumulation of a

large mast cell burden in vivo is usually not observed. Thus, a

homeostatic mechanism must exist to limit differentiation and

accumulation of mast cells in peripheral tissues, both during basal

maintenance, and during mast cell hyperplasia in inflammatory

processes [36]. MiR-221 is a likely candidate as a regulator of mast

cell functions: we previously showed that it is transcriptionally

induced upon mast cell activation, and that it contributes to the

modulation of proliferation in unstimulated mast cells [8]. We now

showed that miR-221 may have more ubiquitous effects to fine-

Figure 5. MiR-221 influences BMMCs actin cytoskeleton. A) Transduced BMMCs were loaded with CFSE (green) and allowed to adhere to amonolayer of 3T3 cells. After washing to remove non-adherent cells, F-actin was stained with phalloidin-AlexaFluor-594 (red) prior imaging with afluorescence microscope. Shown is one representative experiment out of three. B) BMMCs were transduced with the indicated miRT vectors, andoverall F-Actin content was assessed by FACS staining with phalloidin. Shown is one representative experiment out of three.doi:10.1371/journal.pone.0026133.g005



tune proliferation and actin cytoskeleton in cells as different as

resting mast cells and fibroblasts. On the other hand, miR-221 also

influenced many other features of differentiated mast cells,

including cytokine production, migration, adhesion and survival

upon withdrawal of essential cytokines (Supplementary Figure S3),

all mechanisms that may be involved in regulating tissue

accumulation and resolution of hyperplasia upon eradication of

inflammation in vivo.

While we were able to show that at least some of these effects

may be linked to a miR-221-dependent regulation of the actin

cytoskeleton in mast cells, this miRNA may have both ‘house-

keeping’ functions, active in different cell types, like the regulation

of the cytoskeleton and cell cycle, but also cell-specific effects on

targets such as KIT and CD25 that are expressed by mast cells but

not fibroblasts. As for targets, our arrays data and bioinformatics

analysis showed that expression of miR-221 led to the downreg-

ulation of 343 genes, many of which are likely to be primary

targets, containing the miR-221 seed-complementary sequence in

their 39UTR. For this reason, we think that the effect of miR-221

in resting mast cells may be composite and due to small alterations

of many genes in the transcriptome. However, since we did not

perform knockdown of every individual potential target, the

possibility remains that the effect of miR-221 in mast cells and

fibroblasts might be mediated by the downregulation of one or few

predominant genes that we were so far unable to identify. Yet,

although levels of p27Kip1 in miR-221 overexpressing fibroblasts

were very low, the individual knockdown of this protein led to a

phenotype that somewhat resembled the phenotype of p27Kip1-

deleted fibroblasts, but did not resemble in any way the phenotype

of miR-221 overexpressing cells, suggesting that miR-221 target

regulation is more complex then the downregulation of one

predominant target.

While it is clear that miRNAs regulate many different cellular

processes, understanding the details of the functions of individual

miRNAs remains challenging. Indeed, as in the case of miR-221

and -222, miRNAs are frequently present as families of redundant

genes. MiR-221 and -222 share the same seed sequence (Figure 1B),

and should recognize the same targets [9]; although most of the

experiments shown here were performed primarily with miR-221,

we also performed experiments using miR-222-expressing vectors,

which always gave results similar to miR-221, both in mast cells and

in fibroblasts (not shown). Another difficult aspect of dealing with

miRNAs is that each miRNA has many potential targets with

disparate functions, with no means to decide a priori which is the

most meaningful and therefore worthy of experimental validation.

Finally, the degree of target downregulation is typically less than

50% [29,37], and understanding which fraction of the miRNA:-

target interaction is actually relevant for a biological response

remains a challenging task. In our hands, p27Kip1 was a striking

example of this phenomenon, as while it was downregulated very

strongly upon miR-221 expression, its knockdown did not

recapitulate the complex effect of miR-221 expression.

Figure 6. MiR-221 affects cell proliferation and actin cytoskeleton in fibroblasts. A) 3T3 cells were transduced with lentiviral vectors toexpress either miR-221 or controls shLuc and miR-221m. After selection with 2 mg/mL for at least 2 days, total RNA was extracted and levels of miR-221 expression were assessed by TaqMan qRT-PCR. SnoRNA202 was used as endogenous control; for comparison, levels of miR-221 expression in theshLuc-transduced sample were set to one. Representative of two independent experiments. B) 3T3 cells as in A) were lysed in Laemmli sample bufferand p27Kip1 expression was analyzed by Western blot. Expression of b-tubulin was used for normalization in the quantification of bands intensity(numbers below the blot). One representative experiment out of 3 is shown. C) Cells as in A) were collected during exponential-phase growth and theDNA content was assessed with propidium iodide staining as a measure of the number of cells in each stage of the cell cycle. For simplicity, only thepercentage of G1 cells (left in each plot) versus the percentage of dividing cells (S+G2+M) are shown. One out of three independent experiments isshown. D) Cells as in A) were seeded on a coverslip and allowed to adhere overnight, after which they were fixed in 4% paraformaldehyde andstained with phalloidin-AlexaFluor-594 prior imaging with a fluorescent microscope. Shown is one representative experiment out of two. E) Cells as inA) were trypsinized to single cell suspension, fixed and stained as in B) and analyzed by FACS. The left plot shows (on a log scale) the unstainedcontrol compared to stained samples, while on the left the scale was changed to linear to better show the differences among samples. The MFI foreach sample is also provided. Depending on the experiment, control cells were either untransduced cells, cells transduced with shLuc- or the miR-221m-expressing vector, or all of the above. Shown is one representative experiment out of four.doi:10.1371/journal.pone.0026133.g006



Although clearly able to regulate some general cellular features,

such as cell cycle and cytoskeleton in resting mast cell, we

speculate that miR-221/-222 may also be part of a mechanism

that contribute to mediate many of the changes that occur in mast

cells upon stimulation. Specifically, miR-221 may have a

‘housekeeping’ function in resting mast cells, where it is expressed

at low, basal levels, and contributes to the regulation of the cell

cycle and cytoskeleton. Vice versa, miR-221 is also transcription-

ally activated upon stimulation, and in this case it would contribute

to the regulation of cell-type specific, FceRI-dependent mecha-

nisms, such as cytokine production, degranulation and cell

adhesion. In this perspective, it is important to highlight that

although transcription of pri-miR-221-222 starts early upon IgE

stimulation, mature miR-221 accumulation occurs with a ‘slow’

kinetic (the peak of mature miRNA expression is reached at ,24h

after initial stimulation) [8]. We therefore speculate that the slow

kinetic of accumulation of the mature form of miR-221 may

actually contribute to a resting, but ‘activation-ready’ cellular state,

that favors increased degranulation, adherence and cytokine

production upon challenge (Supplementary Figure S4).

Indeed, mast cell stimulation normally leads to increased

adherence and increased survival of the cells, which can be

further activated by a secondary challenge [26,38]. Upon

secondary encounter with the antigen mast cells respond with

increased degranulation and cytokine production (reviewed in

[38]). Although such higher levels of degranulation and cytokine

production are usually considered to be the result of increased

levels of FceRI, the fact that miR-221-expressing cells showed no

perturbation of FceRI expression and at the same time increased

adherence (even in the absence of stimulation), increased

degranulation as well as cytokine production suggests that miR-

221 may contribute to such intensified cellular response upon

secondary challenge. The processes mediated by this miRNA may

also have a role in regulating mast cell homeostasis in tissues and

possibly also in pathologic conditions. It will be therefore

interesting to assess what is the role of miR-221/-222 in vivo in

mouse models, both in terms of cell homeostasis at steady-state or

upon sensitization and challenge with an antigen or with a

pathogen, and this will be the subject of future work.

Along the line of a possible role of these miRNAs in mast cell-

related diseases, we showed that miR-221 and miR-222 regulate

levels of KIT expression in mast cells. Of note, KIT levels are

often abnormally low in patients with indolent systemic mastocy-

tosis, although the D816V gain-of-function mutation in KIT is

Figure 7. Knockdown of the miR-221 target p27Kip1 does not recapitulate the composite effects of miR-221 expression. A) 3T3 cellswere transfected with siRNA oligonucleotides against p27Kip1 (si p27) or with a non-targeting control (si CTRL). 48–72 h later cells were collected andlysed for Western blot analysis. Shown is the quantification (against b-tubulin, used as loading control) of one representative blot; the actual blot forp27Kip1 is shown in the inset. Efficiency of transfection ranged between 50–70% depending on the experiment, with a downregulation of proteinexpression as assessed by Western blot that ranged widely between 0.05–80% of the levels of control, with the latter case (highest expression,minimal knockdown) showing no phenotypic effect. B) 3T3 cells as in A) were trypsinized to single-cell suspension, fixed in ethanol and stained withpropidium iodide for cell-cycle analysis. C) Cells as in A) were seeded on coverslips and allowed to adhere overnight prior fixation with 4%paraformaldehyde and staining with phalloidin-AlexaFluor-594. Nuclei were counterstained with DAPI (blue). D) Quantification of the percentage of‘small, round’ cells observed in an experiment as in C). For each sample, 8 random fields were counted, at a magnification of 10x.doi:10.1371/journal.pone.0026133.g007



present in most of these patients and is sufficient to cause indolent

mastocytosis [39]. This downregulation of KIT expression is even

more pronounced in poor prognosis systemic mastocytosis (i.e.

systemic mastocytosis associated with a clonal hematopoietic

disease, aggressive systemic mastocytosis and mast cell leukemia)

[39], suggesting that multiple layers of regulation of this receptor

may be acting in diseased cells. In this context, it will be interesting

to assess whether miR-221/-222 could be part of a molecular

mechanism involved in KIT regulation specifically in mastocytosis.

Indeed, although the D816V mutation may be sufficient to cause

indolent mastocytosis, other additional defects, that remain to be

identified, are required to induce aggressive mast cell disorders,

and may very well include dysregulated miRNA expression

[40,41,42]. This is even more intriguing considering that CD25,

which is so far the best available diagnostic marker for systemic

mastocytosis with bone marrow involvement [32,33], is also

regulated by these miRNAs. While miR-221/-222 were already

implicated in various human cancers for their effect on

proliferation, we show here for the first time that these miRNAs

also regulate mast cell adhesion, migration, and survival, all

processes that may have implications in mastocytosis.

In summary, although miR-221 doesn’t seem to affect mast cell

differentiation, it influences many features of the biology of

differentiated cells. Specifically, at basal levels, in resting

conditions, these effects are likely to be linked, at least in part,

to the regulation of the actin cytoskeleton and cell cycle, two

features that are regulated by miR-221 independently of the cell

type. However, upon mast cell stimulation, miR-221 may have

some more cell type-specific, activation-dependent effects, influ-

encing the extent of degranulation, adherence and cytokine

production in response to IgE-antigen complexes. Although still

speculative, we propose a model in which miR-221 would have

two different roles in mast cells: in resting cells, it contributes to

normal cell homeostasis through the regulation of the cell cycle

and cytoskeleton, while upon induction following acute stimula-

tion, it contributes to increase the strength of the response to

antigenic challenge. Overall, our work provides new insights into

previously unknown effects of miR-221 in mast cell biology, and

may have important implications for our understanding of the

molecular mechanisms underlying normal and pathologic mast

cell conditions.

Supporting Information

Figure S1 MiR-221 expression can be regulated by thetranscriptional repressor PLZF, but it has no role inBMMC differentiation. A) Lineage depleted (Lin–, lacking

surface expression of CD5, CD45R, CD11b, Gr-1, 7-4 and Ter-

119) and Lin+ bone marrow cells were either immediately used for

RNA extraction or differentiated to mast cells in IL-3 containing

medium [43]. Total RNA from Lin– derived mast cells was used to

assess Plzf mRNA expression (upper panel) and miR-221 (lower panel).

B) Differentiated BMMCs were lentivirally transduced to

ectopically express PLZF. After puromycin selection for 48h, cells

were either left untreated or were stimulated with 20nM PMA and

1 mM ionomycin for 24h, prior RNA extraction and analysis of

Plzf and miR-221 expression. C) Lin– cells were transduced with

the indicated vectors to either force (pAPM) or ablate (miRT)

miR-221 expression, and were cultured for three weeks in the

presence of IL-3 to allow mast cell differentiation. Cultures were

analyzed weekly for the presence of mast cells (FceRIa+ KIT+) by

surface staining. Each point represents one independent experi-

ment. Cells transduced with shLuc, miR-221 and miR-221m

vectors were selected with 2 mg/mL puromycin, while cells

transduced with the miRT vectors (empty, T-221 and T-221-

222) were FACS-sorted for GFP expression. D) Total RNA was

extracted from cells treated as in C) at the end of the

differentiation period (percentage of FceRIa+ KIT+ cells was

greater than 90%), and expression of miR-221 was assessed by

TaqMan qRT-PCR. SnoRNA202 was used as endogenous

control, with levels of miR-221 expression set to one in the

shLuc-transduced sample. Cells transduced with shLuc and miR-

221 vectors were selected with 2 mg/mL puromycin, while cells

transduced with the miRT vectors (empty, T-221 and T-221-222)

were FACS-sorted for GFP expression.

(TIF)

Figure S2 MiR-221 expression does not significantlyalter ERK phosphorylation in mast cells, but favorscytokine production. A) Differentiated BMMCs transduced

with the indicated vectors were sensitized with 1.5 mg/mL of IgE-

anti-DNP for 15min on ice. After washing to remove unbound

IgE, 200ng/mL of DNP-HSA were added, and the cells were

immediately moved to a 37uC water bath for 5, 15 and 45min.

Cells were subsequently fixed, permeabilized and stained with

biotinylated anti-phospho-p44/42 MAPK (Erk1/2). The mean

fluorescence intensity for each sample is indicated on the left. B)

Cells as in A) were either left untreated or were stimulated for

5min with 1 mM ionomycin and 20nM PMA at 37uC, after which

cells were fixed, permeabilized and stained with an anti-Erk1/2

antibody. The mean fluorescence intensity for each sample is

indicated next to the histograms. Shown is one experiment out of

two. C) BMMCs as in A) were stimulated with 1.5 mg/mL IgE-

anti-DNP and 200ng/mL DNP-HSA for 3.5h at 37uC. To block

export from the Golgi, brefeldin A (10 mg/mL) was added in the

last two hours of stimulation. Cells were subsequently fixed,

permeabilized and stained with anti-IL-6-PE and anti-TNF-a-PE-

Cy7. One representative experiment out of three is shown.

(TIF)

Figure S3 MiR-221 expression favors mast cell survivalin response to withdrawal of essential cytokines. BMMCs

were transduced with the indicated vectors and differentiated in

the presence of IL-3 only (top panel) or IL-3 + 10ng/mL SCF (bottom

panel) for three weeks, after which all cytokines were washed out of

the culture medium for at least 24h prior evaluation of early cell

death with annexin V and 7AAD staining. Shown is the

percentage of cells in early apoptosis (annexin V+ 7AAD–).

(TIF)

Figure S4 A ‘dual’ role for miR-221 in mast cells.Speculative model of the possible roles of miR-221 in mast cells.

At resting state, basal levels of miR-221 expression would regulate

homeostatic mechanisms such as the cell cycle and cytoskeleton.

These effects are not necessarily cell type-specific, as they can be

active also in fibroblasts, which also express miR-221. Upon mast

cell activation, ‘early’ effects include the release of preformed

mediators from the cytoplasmic granules and the de novo synthesis

of other mediators, including a broad panel of cytokines. The peak

of accumulation of mature miR-221 is instead a ‘late’ event upon

cell stimulation, and we speculate that it may contribute to the

strength of the response upon secondary challenge, with increased

degranulation, cytokine production and cell adherence.

(TIF)

Methods S1

(DOC)



Acknowledgments

We thank Anjana Rao and Federica Sallusto for critical reading of the

manuscript. Special thanks also to Michele Proietti, Fabio Grassi,

Mariagrazia Uguccioni and Marcus Thelen for sharing reagents.

Author Contributions

Conceived and designed the experiments: SM. Performed the experiments:

RJM LD NR SM. Analyzed the data: SM. Wrote the paper: SM.

Performed Sylamer analysis: NB HKS AJE.

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